Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies

Rong Xu, Brenda Ayers, David Cowburn, Tom W. Muir

Research output: Contribution to journalArticle

194 Citations (Scopus)

Abstract

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Original languageEnglish (US)
Pages (from-to)388-393
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number2
DOIs
StatePublished - Jan 19 1999
Externally publishedYes

Fingerprint

Recombinant Proteins
Ligation
src Homology Domains
Protein-Tyrosine Kinases
Proteins
Product Labeling
Protein Folding
Mass Spectrometry
Ligands
Peptides
Mutation
In Vitro Techniques
Protein Domains

Keywords

  • Expressed protein ligation
  • Segmental labeling
  • Src homology domains
  • Structural biology

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Chemical ligation of folded recombinant proteins : Segmental isotopic labeling of domains for NMR studies. / Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 2, 19.01.1999, p. 388-393.

Research output: Contribution to journalArticle

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